Probiotics are suggested to improve pig health, nutrient utilisation, performance, and may reduce nitrogen (N) pollution. However, the effectivity of a single-strain might be different from a multi-strain. The study was conducted to investigate the effect of a novel Bacillus multi-strain on nutrient digestibility, energy utilisation, and N retention in weaned piglets. The experiment consisted of a control diet (CD) and a supplemented diet (SD). The probiotic used for SD consisted of B. amyloliquefaciens - 516 and B. subtilis - 541. A total of 8 boars/ treatment were weaned (day 0; 8.5 kg bodyweight). Until day 10, piglets were fed ad libitum and were housed in pairs; from day 11, piglets were fed semi ad libitum and were housed individually. From day 14, faecal and urine were collected twice daily. Piglets were humanely euthanised at day 19 (15.0 kg bodyweight) after which the jejunum, ileum, and colon content were collected. In faeces the apparent total tract digestibility (ATTD) of, amongst others, DM, organic matter (OM), crude protein (CP), non-starch polysaccharides (NSP), and subsequently net energy (NE) were calculated using titanium dioxide as an indigestible marker. In jejunum and ileum, the apparent digestibility of CP was estimated and in ileum the apparent AA digestibility. In urine, N content was measured to determine N retention. The volatile fatty acid (VFA), branched-chain fatty acid (BCFA), and lactic acid content were analysed in colon and faeces. Apparent CP digestibility in jejunum and ileum were not affected by treatment (P>0.05) and no effect was observed on apparent ileal digestibility of AA (P>0.05). Supplementation with the multi-strain probiotic improved ATTD of DM (P=0.01; +1.3%) and OM (P=0.02; +1.2%) and tended to improved ATTD of CP (P=0.10; +2.2%) and NSP (P=0.07; +1.9%). The multi-strain probiotic also improved the NE value (P=0.02; +0.2 MJ/kg DM) and improved N retention (P=0.05; +1.6%). Supplementation did not influence VFA, BCFA, and lactic acid content in the faeces (P>0.05). However, in colon, supplementation did influence lactic acid content (lower; P=0.01) and tended to influence valeric acid content (higher; P=0.09). In conclusion, results from the current study suggests that the multi-strain probiotic has the potential to contribute to improve nutrient efficiency in weaned piglets. More research needs to be done to identify the impact of the improved nutrient utilization on gut health in post-weaned pigs as well as environmental pollution.